ABSTRACTThe role of Foxp3-expressing regulatory T (T(reg)) cells in tolerance and autoimmunity is well-established. However, although of considerable clinical interest, the role of T(reg) cells in the regulation of hematopoietic homeostasis remains poorly understood. Thus, we analysed B and T lymphopoiesis in the scurfy (Sf) mouse model of T(reg) cell deficiency. In these experiments, the near-complete block of B lymphopoiesis in the BM of adolescent Sf mice was attributed to autoimmune T cells. We could exclude a constitutive lympho-hematopoietic defect or a B cell-intrinsic function of Foxp3. Efficient B cell development in the BM early in ontogeny and pronounced extramedullary B lymphopoietic activity resulted in a peripheral pool of mature B cells in adolescent Sf mice. However, marginal zone B and B-1a cells were absent throughout ontogeny. Developmental B lymphopoietic defects largely correlated with defective thymopoiesis. Importantly, neonatal adoptive T(reg) cell therapy suppressed exacerbated production of inflammatory cytokines and restored thymopoiesis but was ineffective in recovering defective B lymphopoiesis, probably due to a failure to compensate production of stroma cell-derived IL-7 and CXCL12. Our observations on autoimmune-mediated incapacitation of the BM environment in Foxp3-deficient mice will have direct implications for the rational design of BM transplantation protocols for patients with severe genetic deficiencies in functional Foxp3(+) T(reg) cells.

Mentions:
We initially focused on the assessment of B and T lymphopoietic activity in adolescent Sf mice. In this context, it is important to note that all Sf mice in our colony housed under specific pathogen-free conditions develop external signs of severe autoimmune disease (general failure to thrive, exfoliative dermatitis, etc.) by 4 weeks of age (data not shown), with a median survival of 27 days (see also Figure 5A). Flow cytometric analysis of moribund 4-week-old Sf mice revealed an up to 10-fold reduction in both percentages and numbers of early B220+c-kit+ Pro/Pre-B-I cells (nomenclature according to Rolink et al., 1999), as compared to age-matched wild-type (WT) control animals (Figure 1A, left). In WT mice, developmental progression of Pre-B-I cells gives rise to a pronounced compartment of B220+CD25+ Pre-B-II cells, which ultimately generate substantial populations of immature B220lowsIgM+ B cells (Figure 1A, top). In contrast, in Sf mice, regardless of the existence of a clearly discernable, albeit dramatically reduced population of Pro/Pre-B-I cells, subsequent Pre-B-II and immature sIgM+ B cell stages were consistently found to be below the level of detection (Figure 1A, middle and right). Similar results were obtained with adolescent Foxp3ΔEGFP mice (data not shown) that lack functional Treg cells due to a targeted insertion of GFP-encoding sequence into the Foxp3 locus, resulting in the expression of a non-functional Foxp3 protein fused to GFP (Lin et al., 2007). Overall, these data are largely consistent with previous reports (Leonardo et al., 2010; Chang et al., 2012) in that B cell development in the BM of adolescent Foxp3-deficient mice is severely abrogated.

Mentions:
We initially focused on the assessment of B and T lymphopoietic activity in adolescent Sf mice. In this context, it is important to note that all Sf mice in our colony housed under specific pathogen-free conditions develop external signs of severe autoimmune disease (general failure to thrive, exfoliative dermatitis, etc.) by 4 weeks of age (data not shown), with a median survival of 27 days (see also Figure 5A). Flow cytometric analysis of moribund 4-week-old Sf mice revealed an up to 10-fold reduction in both percentages and numbers of early B220+c-kit+ Pro/Pre-B-I cells (nomenclature according to Rolink et al., 1999), as compared to age-matched wild-type (WT) control animals (Figure 1A, left). In WT mice, developmental progression of Pre-B-I cells gives rise to a pronounced compartment of B220+CD25+ Pre-B-II cells, which ultimately generate substantial populations of immature B220lowsIgM+ B cells (Figure 1A, top). In contrast, in Sf mice, regardless of the existence of a clearly discernable, albeit dramatically reduced population of Pro/Pre-B-I cells, subsequent Pre-B-II and immature sIgM+ B cell stages were consistently found to be below the level of detection (Figure 1A, middle and right). Similar results were obtained with adolescent Foxp3ΔEGFP mice (data not shown) that lack functional Treg cells due to a targeted insertion of GFP-encoding sequence into the Foxp3 locus, resulting in the expression of a non-functional Foxp3 protein fused to GFP (Lin et al., 2007). Overall, these data are largely consistent with previous reports (Leonardo et al., 2010; Chang et al., 2012) in that B cell development in the BM of adolescent Foxp3-deficient mice is severely abrogated.

Bottom Line:
However, marginal zone B and B-1a cells were absent throughout ontogeny.Developmental B lymphopoietic defects largely correlated with defective thymopoiesis.Importantly, neonatal adoptive T(reg) cell therapy suppressed exacerbated production of inflammatory cytokines and restored thymopoiesis but was ineffective in recovering defective B lymphopoiesis, probably due to a failure to compensate production of stroma cell-derived IL-7 and CXCL12.

ABSTRACTThe role of Foxp3-expressing regulatory T (T(reg)) cells in tolerance and autoimmunity is well-established. However, although of considerable clinical interest, the role of T(reg) cells in the regulation of hematopoietic homeostasis remains poorly understood. Thus, we analysed B and T lymphopoiesis in the scurfy (Sf) mouse model of T(reg) cell deficiency. In these experiments, the near-complete block of B lymphopoiesis in the BM of adolescent Sf mice was attributed to autoimmune T cells. We could exclude a constitutive lympho-hematopoietic defect or a B cell-intrinsic function of Foxp3. Efficient B cell development in the BM early in ontogeny and pronounced extramedullary B lymphopoietic activity resulted in a peripheral pool of mature B cells in adolescent Sf mice. However, marginal zone B and B-1a cells were absent throughout ontogeny. Developmental B lymphopoietic defects largely correlated with defective thymopoiesis. Importantly, neonatal adoptive T(reg) cell therapy suppressed exacerbated production of inflammatory cytokines and restored thymopoiesis but was ineffective in recovering defective B lymphopoiesis, probably due to a failure to compensate production of stroma cell-derived IL-7 and CXCL12. Our observations on autoimmune-mediated incapacitation of the BM environment in Foxp3-deficient mice will have direct implications for the rational design of BM transplantation protocols for patients with severe genetic deficiencies in functional Foxp3(+) T(reg) cells.